Leucine-based motif and clostridial neurotoxins

ABSTRACT

Modified neurotoxin comprising neurotoxin including structural modification, wherein the structural modification alters the biological persistence, preferably the biological half-life, of the modified neurotoxin relative to an identical neurotoxin without the structural modification. The structural modification includes addition or deletion of a leucine-based motif or parts thereof. In one embodiment, methods of making the modified neurotoxin include using recombinant techniques. In another embodiment, methods of using the modified neurotoxin to treat biological disorders include treating autonomic disorders, neuromuscular disorders or pains.

This application is a divisional and claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 11/039,268, filed Jan.19, 2005, now U.S. Pat. No. 7,393,925 an divisional application thatclaims priority pursuant to 35 U.S.C. §120 to U.S. patent applicationSer. No. 09/620,840, filed Jul. 21, 2000, now U.S. Pat. No. 6,903,187each of which is hereby incorporated by reference in its entirety.

BACKGROUND

The present invention relates to modified neurotoxins, particularlymodified Clostridial neurotoxins, and use thereof to treat variousdisorders, including neuromuscular disorders, autonomic nervous systemdisorders and pain.

The clinical use of botulinum toxin serotype A (herein after “BoNT/A”),a serotype of Clostridial neurotoxin, represents one of the mostdramatic role reversals in modern medicine: a potent biologic toxintransformed into a therapeutic agent. BoNT/A has become a versatile toolin the treatment of a wide variety of disorders and conditionscharacterized by muscle hyperactivity, autonomic nervous systemhyperactivity and/or pain.

Botulinum Toxin

The anaerobic, gram positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin, botulinum toxin, which causes aneuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

BoNT/A is the most lethal natural biological agent known to man. About50 picograms of botulinum toxin (purified neurotoxin complex) serotype Ais a LD₅₀ in mice. One unit (U) of botulinum toxin is defined as theLD₅₀ upon intraperitoneal injection into female Swiss Webster miceweighing 18-20 grams each. Seven immunologically distinct botulinumneurotoxins have been characterized, these being respectively botulinumneurotoxin serotypes A, B, C₁, D, E, F and G each of which isdistinguished by neutralization with serotype-specific antibodies. Thedifferent serotypes of botulinum toxin vary in the animal species thatthey affect and in the severity and duration of the paralysis theyevoke. For example, it has been determined that BoNt/A is 500 times morepotent, as measured by the rate of paralysis produced in the rat, thanis botulinum toxin serotype B (BoNT/B). Additionally, BoNt/B has beendetermined to be non-toxic in primates at a dose of 480 U/kg which isabout 12 times the primate LD₅₀ for BoNt/A. Botulinum toxin apparentlybinds with high affinity to cholinergic motor neurons, is translocatedinto the neuron and blocks the release of acetylcholine.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletalmuscles. BoNt/A has been approved by the U.S. Food and DrugAdministration for the treatment of blepharospasm, strabismus andhemifacial spasm. Non-serotype A botulinum toxin serotypes apparentlyhave a lower potency and/or a shorter duration of activity as comparedto BoNt/A. Clinical effects of peripheral intramuscular BoNt/A areusually seen within one week of injection. The typical duration ofsymptomatic relief from a single intramuscular injection of BoNt/Aaverages about three months.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. For example, botulinumserotypes A and E both cleave the 25 kiloDalton (kD) synaptosomalassociated protein (SNAP-25), but they target different amino acidsequences within this protein. BoNT/B, D, F and G act onvesicle-associated protein (VAMP, also called synaptobrevin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin serotype C₁ (BoNT/C₁) has been shown to cleave both syntaxin andSNAP-25. These differences in mechanism of action may affect therelative potency and/or duration of action of the various botulinumtoxin serotypes.

Regardless of serotype, the molecular mechanism of toxin intoxicationappears to be similar and to involve at least three steps or stages. Inthe first step of the process, the toxin binds to the presynapticmembrane of the target neuron through a specific interaction between theH chain and a cell surface receptor; the receptor is thought to bedifferent for each serotype of botulinum toxin and for tetanus toxin.The carboxyl end segment of the H chain, H_(c), appears to be importantfor targeting of the toxin to the cell surface.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This last step is thought to be mediated by the amino end segmentof the H chain, H_(N), which triggers a conformational change of thetoxin in response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxinthen translocates through the endosomal membrane into the cytosol.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the H and L chain. Theentire toxic activity of botulinum and tetanus toxins is contained inthe L chain of the holotoxin; the L chain is a zinc (Zn++) endopeptidasewhich selectively cleaves proteins essential for recognition and dockingof neurotransmitter-containing vesicles with the cytoplasmic surface ofthe plasma membrane, and fusion of the vesicles with the plasmamembrane. Tetanus neurotoxin, botulinum toxin/B/D,/F, and/G causedegradation of synaptobrevin (also called vesicle-associated membraneprotein (VAMP)), a synaptosomal membrane protein. Most of the VAMPpresent at the cytosolic surface of the synaptic vesicle is removed as aresult of any one of these cleavage events. Each toxin specificallycleaves a different bond.

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the BoNt/Acomplex can be produced by Clostridial bacterium as 900 kD, 500 kD and300 kD forms. BoNT/B and C₁ are apparently produced as only a 500 kDcomplex. BoNT/D is produced as both 300 kD and 500 kD complexes.Finally, BoNT/E and F are produced as only approximately 300 kDcomplexes. The complexes (i.e. molecular weight greater than about 150kD) are believed to contain a non-toxin hemaglutinin protein and anon-toxin and non-toxic nonhemaglutinin protein. These two non-toxinproteins (which along with the botulinum toxin molecule comprise therelevant neurotoxin complex) may act to provide stability againstdenaturation to the botulinum toxin molecule and protection againstdigestive acids when toxin is ingested. Additionally, it is possiblethat the larger (greater than about 150 kD molecular weight) botulinumtoxin complexes may result in a slower rate of diffusion of thebotulinum toxin away from a site of intramuscular injection of abotulinum toxin complex.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP and glutamate.

BoNt/A can be obtained by establishing and growing cultures ofClostridium botulinum in a fermenter and then harvesting and purifyingthe fermented mixture in accordance with known procedures. All thebotulinum toxin serotypes are initially synthesized as inactive singlechain proteins which must be cleaved or nicked by proteases to becomeneuroactive. The bacterial strains that make botulinum toxin serotypes Aand G possess endogenous proteases and serotypes A and G can thereforebe recovered from bacterial cultures in predominantly their active form.In contrast, botulinum toxin serotypes C₁, D and E are synthesized bynonproteolytic strains and are therefore typically unactivated whenrecovered from culture. Serotypes B and F are produced by bothproteolytic and nonproteolytic strains and therefore can be recovered ineither the active or inactive form. However, even the proteolyticstrains that produce, for example, the BoNt/B serotype only cleave aportion of the toxin produced. The exact proportion of nicked tounnicked molecules depends on the length of incubation and thetemperature of the culture. Therefore, a certain percentage of anypreparation of, for example, the BoNt/B toxin is likely to be inactive,possibly accounting for the known significantly lower potency of BoNt/Bas compared to BoNt/A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that BoNt/B has, upon intramuscular injection,a shorter duration of activity and is also less potent than BoNt/A atthe same dose level.

It has been reported that BoNt/A has been used in clinical settings asfollows:

(1) about 75-125 units of BOTOX®¹ per intramuscular injection (multiplemuscles) to treat cervical dystonia; ¹Available from Allergan, Inc., ofIrvine, Calif. under the tradename BOTOX®.

(2) 5-10 units of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) (5 units injected intramuscularly into the procerusmuscle and 10 units injected intramuscularly into each corrugatorsupercilii muscle);

(3) about 30-80 units of BOTOX® to treat constipation by intrasphincterinjection of the puborectalis muscle;

(4) about 1-5 units per muscle of intramuscularly injected BOTOX® totreat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

(5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

(6) to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows:

-   -   (a) flexor digitorum profundus: 7.5 U to 30 U    -   (b) flexor digitorum sublimus: 7.5 U to 30 U    -   (c) flexor carpi ulnaris: 10 U to 40 U    -   (d) flexor carpi radialis: 15 U to 60 U    -   (e) biceps brachii: 50 U to 200 U. Each of the five indicated        muscles has been injected at the same treatment session, so that        the patient receives from 90 U to 360 U of upper limb flexor        muscle BOTOX® by intramuscular injection at each treatment        session.

The success of BoNt/A to treat a variety of clinical conditions has ledto interest in other botulinum toxin serotypes. A study of twocommercially available BoNT/A preparations (BOTOX® and Dysport®) andpreparations of BoNT/B and F (both obtained from Wako Chemicals, Japan)has been carried out to determine local muscle weakening efficacy,safety and antigenic potential. Botulinum toxin preparations wereinjected into the head of the right gastrocnemius muscle (0.5 to 200.0units/kg) and muscle weakness was assessed using the mouse digitabduction scoring assay (DAS). ED₅₀ values were calculated from doseresponse curves. Additional mice were given intramuscular injections todetermine LD₅₀ doses. The therapeutic index was calculated as LD₅₀/ED₅₀.Separate groups of mice received hind limb injections of BOTOX® (5.0 to10.0 units/kg) or BoNt/B (50.0 to 400.0 units/kg), and were tested formuscle weakness and increased water consumption, the later being aputative model for dry mouth. Antigenic potential was assessed bymonthly intramuscular injections in rabbits (1.5 or 6.5 ng/kg for BoNt/Bor 0.15 ng/kg for BOTOX®). Peak muscle weakness and duration were doserelated for all serotypes. DAS ED₅₀ values (units/kg) were as follows:BOTOX®: 6.7, Dysport®: 24.7, BoNt/B: 27.0 to 244.0, BoNT/F: 4.3. BOTOX®had a longer duration of action than BoNt/B or BoNt/F. Therapeutic indexvalues were as follows: BOTOX®: 10.5, Dysport®: 6.3, BoNt/B: 3.2. Waterconsumption was greater in mice injected with BoNt/B than with BOTOX®,although BoNt/B was less effective at weakening muscles. After fourmonths of injections 2 of 4 (where treated with 1.5 ng/kg) and 4 of 4(where treated with 6.5 ng/kg) rabbits developed antibodies againstBoNt/B. In a separate study, 0 of 9 BOTOX® treated rabbits demonstratedantibodies against BoNt/A. DAS results indicate relative peak potenciesof BoNt/A being equal to BoNt/F, and BoNt/F being greater than BoNt/B.With regard to duration of effect, BoNt/A was greater than BoNt/B, andBoNt/B duration of effect was greater than BoNt/F. As shown by thetherapeutic index values, the two commercial preparations of BoNt/A(BOTOX® and Dysport®) are different. The increased water consumptionbehavior observed following hind limb injection of BoNt/B indicates thatclinically significant amounts of this serotype entered the murinesystemic circulation. The results also indicate that in order to achieveefficacy comparable to BoNt/A, it is necessary to increase doses of theother serotypes examined. Increased dosage can comprise safety.Furthermore, in rabbits, serotype B was more antigenic than was BOTOX®,possibly because of the higher protein load injected to achieve aneffective dose of BoNt/B.

The tetanus neurotoxin acts mainly in the central nervous system, whilebotulinum neurotoxin acts at the neuromuscular junction; both act byinhibiting acetylcholine release from the axon of the affected neuroninto the synapse, resulting in paralysis. The effect of intoxication onthe affected neuron is long-lasting and until recently has been thoughtto be irreversible. The tetanus neurotoxin is known to exist in oneimmunologically distinct serotype.

Acetylcholine

Typically only a single type of small molecule neurotransmitter isreleased by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsympathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic and most of the postganglionicneurons of the sympathetic nervous system secret the neurotransmitternorepinephrine. In most instances acetylcholine has an excitatoryeffect. However, acetylcholine is known to have inhibitory effects atsome of the peripheral parasympathetic nerve endings, such as inhibitionof the heart by the vagal nerve.

The efferent signals of the autonomic nervous system are transmitted tothe body through either the sympathetic nervous system or theparasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

Acetylcholine activates two types of receptors, muscarinic and nicotinicreceptors. The muscarinic receptors are found in all effector cellsstimulated by the postganglionic neurons of the parasympathetic nervoussystem, as well as in those stimulated by the postganglionic cholinergicneurons of the sympathetic nervous system. The nicotinic receptors arefound in the synapses between the preganglionic and postganglionicneurons of both the sympathetic and parasympathetic. The nicotinicreceptors are also present in many membranes of skeletal muscle fibersat the neuromuscular junction.

Acetylcholine is released from cholinergic neurons when small, clear,intracellular vesicles fuse with the presynaptic neuronal cell membrane.A wide variety of non-neuronal secretory cells, such as, adrenal medulla(as well as the PC12 cell line) and pancreatic islet cells releasecatecholamines and insulin, respectively, from large dense-corevesicles. The PC12 cell line is a clone of rat pheochromocytoma cellsextensively used as a tissue culture model for studies ofsympathoadrenal development. Botulinum toxin inhibits the release ofboth types of compounds from both types of cells in vitro, permeabilized(as by electroporation) or by direct injection of the toxin into thedenervated cell. Botulinum toxin is also known to block release of theneurotransmitter glutamate from cortical synaptosomes cell cultures.

Sanders et al. in U.S. Pat. No. 5,766,605 disclose that BoNT/A can beused to treat autonomic nervous system disorders, for examplerhinorrhea, otitis media, excessive salivation, asthma, chronicobstructive pulmonary disease (COPD), excessive stomach acid secretion,spastic colitis and excessive sweating.

Furthermore, Binder U.S. Pat. No. 5,714,468 discloses that BoNT/A can beused to treat migraine headache pain that is associated with musclespasm, vascular disturbances, neuralgia and neuropathy. Additionally,our laboratory data obtained from experiments with rats show that pain,particularly inflammation pain, may be reduced with an injection ofbotulinum serotype A, either spinally or peripherally.

One of the reasons that BoNT/A has been selected over the otherserotypes, for example serotypes B, C₁, D, E, F, and G, for clinical useis that BoNT/A has a substantially longer lasting therapeutic effect. Inother words, the inhibitory effect of BoNT/A is more persistent.Therefore, the other serotypes of botulinum toxins could potentially beeffectively used in a clinical environment if their biologicalpersistence could be enhanced. For example, parotid sialocele is acondition where the patient suffers from excessive salivation. Sanderset al. disclose in their patent that serotype D may be very effective inreducing excessive salivation. However, the biological persistence ofserotype D botulinum toxin is relatively short and thus may not bepractical for clinical use. If the biological persistence of serotype Dmay be enhanced, it may effectively be used in a clinical environment totreat, for example, parotid sialocele.

Another reason that BoNT/A has been a preferred neurotoxin for clinicaluse is, as discussed above, its superb ability to immobilize musclesthrough flaccid paralysis. For example, BoNT/A is preferentially used toimmobilize muscles and prevent limb movements after a tendon surgery tofacilitate recovery. However, for some minor tendon surgeries, thehealing time is relatively short. It would be beneficial to have aBoNT/A without the prolonged persistence for use in such circumstancesso that the patient can regain mobility at about the same time theyrecover from the surgery.

There is a need to have modified neurotoxins that are non serotype Abotulinum toxins with enhanced biological persistence and modifiedneurotoxins that are BoNT/A with reduced biological persistence andmethods for preparing such toxins.

SUMMARY

The present invention meets this need and provides for non serotype Abotulinum toxins with enhanced persistence and BoNT/A with reducedpersistence and methods for preparing such toxins.

In one broad embodiment of the invention, a modified neurotoxin isformed from a neurotoxin which includes a structural modification. Thestructural modification is able to alter the biological persistence ofthe neurotoxin. In one embodiment, the structural modification includesfusing a biological persistence enhancing component with a neurotoxin.The biological persistence enhancing component increases the duration ofthe inhibitory effect of the modified neurotoxin intracellularly.Preferably, the biological persistence enhancing component is aleucine-based motif.

Without wishing to be limited by any particular theory or mechanism ofoperation, it is believed that the leucine-based motif enhances thepersistence of a neurotoxin by increasing its biological half-life. Forexample, it is known that BoNT/A has a very long biological persistence.Keller et al., FEBS Letters, 456:137-142 (1999), investigated todetermine whether the persistence of BoNT/A is due to a depressedsynthesis of SNAP-25 to replace the cleaved ones, or is due to thestability of the light chain intracellularly. Keller et al. found thatthe major factor limiting cellular recovery is the prolonged stabilityof toxin, or prolonged half-life.

Furthermore, without wishing to be limited by any particular theory ormechanism of operation, it is believed that the leucine-based motiflocated on the light chain, or the third amino acid sequence region, ofBoNT/A, and not on any other serotypes, is responsible for the prolongedhalf-life of BoNT/A.

A leucine-based motif is often found on the carboxyl termini of severalmembrane receptors and vesicular neurotransmitter transporter and itapparently plays a crucial role in vesicle/membrane trafficking. Liu etal. Trends Cell Biol, 9:356-363 (1999); Tan et al. J Biol Chem,273:17351-17360 (1998); Dietrich et al. J. Cell Bio, 138:271-281 (1997);Shin et al. J Biol Chem, 266:10658-10665 (1991) and Geisler et al. JBiol Chem, 273:21316-21323 (1998). More specifically, the leuine-basedmotif is found in a membrane-proximal, cytoplasmic, carboxylic terminaltail of a membrane-bound receptor or transporter protein. It has beendemonstrated that adaptor proteins that are highly concentrated atclathrin coated pits bind to the leucine-based motif and that disruptionof this motif disrupts endocytosis of the motif-containing protein. Tanet al. J Biol Chem, 273:17351-17360 (1998); Dietrich et al. J. Cell Bio,138:271-281 (1997) and Shin et al. J Biol Chem, 266:10658-10665 (1991).Furthermore, addition of the leucine-based motif to the carboxylterminus of the plasma membrane protein Tac resulted in endocytosis ofthe chimera, suggesting that the motif is sufficient for targetedendocytosis. Tan et al. Supra.

The leucine-based motif located on the light chain of BoNT/A may causethe light chain to localize at the membranes, similarly to howmembrane-bound receptor or transporter protein are localized at themembranes described above. Localization of the light chain to themembrane may protect and preserve the light chain, and the heavy chainif it is still attached, from being removed and/or degraded by theintracellular cleaning processes, thereby rendering it a long biologicalhalf-life. For example, intracellular autophagosomes are responsible forcleaning the cytoplasm by engulfing, and thereafter degrading, freefloating foreign substances in the cytoplasm. Erdal et al. NaunynSchmiedebergs Acrch Pharmacol, 351:67-78 (1995). Since the leucine-basedmotif provides an anchor for the light chain, and the heavy chain if itis still attached, it would be difficult for the autophagosomes toremove and engulf the light chain from the cytoplasm. Thus the lightchain remains in the cytoplasm to continue exerting its inhibitoryeffects on vesicular exocytosis of neurotransmitters.

In another embodiment, the leucine-based motif located on the lightchain of BoNT/A is removed in its entirety or in parts. This modifiedneurotoxin effectively has a shortened the biological persistence.Preferably, this modified neurotoxin has a decreased half-life.

This invention also provide for methods of producing modifiedneurotoxins. Additionally, this invention provide for methods of usingthe modified neurotoxins to treat biological disorders.

DEFINITIONS

Before proceeding to describe the present invention, the followingdefinitions are provided and apply herein.

“Heavy chain” means the heavy chain of a clostridial neurotoxin. Itpreferably has a molecular weight of about 100 kDa and may be referredto herein as H chain or as H.

“H_(N)” means a fragment (preferably having a molecular weight of about50 kDa) derived from the H chain of a Clostridial neurotoxin which isapproximately equivalent to the amino terminal segment of the H chain,or the portion corresponding to that fragment in the intact in the Hchain. It is believed to contain the portion of the natural or wild typeclostridial neurotoxin involved in the translocation of the L chainacross an intracellular endosomal membrane.

“H_(C)” means a fragment (about 50 kDa) derived from the H chain of aclostridial neurotoxin which is approximately equivalent to the carboxylterminal segment of the H chain, or the portion corresponding to thatfragment in the intact H chain. It is believed to be immunogenic and tocontain the portion of the natural or wild type Clostridial neurotoxininvolved in high affinity, presynaptic binding to motor neurons.

“Light chain” means the light chain of a clostridial neurotoxin. Itpreferably has a molecular weight of about 50 kDa, and can be referredto as L chain, L or as the proteolytic domain (amino acid sequence) of aclostridial neurotoxin. The light chain is believed to be effective asan inhibitor of neurotransmitter release when it is released into acytoplasm of a target cell.

“Neurotoxin” means a molecule that is capable of interfering with thefunctions of a neuron. The “neurotoxin” may be naturally occurring orman-made.

“Modified neurotoxin” means a neurotoxin which includes a structuralmodification. In other words, a “modified neurotoxin” is a neurotoxinwhich has been modified by a structural modification. The structuralmodification changes the biological persistence, preferably thebiological half-life, of the modified neurotoxin relative to theneurotoxin from which the modified neurotoxin is made. The modifiedneurotoxin is structurally different from a naturally existingneurotoxin.

“Structural modification” means a physical change to the neurotoxin thatmay be affected by covalently fusing one or more amino acids to theneurotoxin. “Structural modification” also means the deletion of one ormore amino acids from a neurotoxin. Furthermore, “structuralmodification” may also mean any changes to a neurotoxin that makes itphysically or chemically different from an identical neurotoxin withoutthe structural modification.

“Biological persistence” means the time duration in which a neurotoxinor a modified neurotoxin causes an interference with a neuronalfunction, for example the time duration in which a neurotoxin or amodified neurotoxin causes a substantial inhibition of the release ofacetylcholine from a nerve terminal.

“Biological half-life” means the time that the concentration of aneurotoxin or a modified neurotoxin, preferably the active portion ofthe neurotoxin or modified neurotoxin, for example the light chain ofbotulinum toxins, is reduced to half of the original concentration in amammal, preferably in the neurons of the mammal.

DETAILED DESCRIPTION

The present invention is based upon the discovery that the biologicalpersistence of a neurotoxin may be altered by structurally modifying theneurotoxin. In other words, a modified neurotoxin with an alteredbiological persistence may be formed from a neurotoxin containing orincluding a structural modification. In one embodiment, the structuralmodification includes the fusing a biological persistence enhancingcomponent to the primary structure of a neurotoxin to enhance itsbiological persistence. In a preferred embodiment, the biologicalpersistence enhancing component is a leucine-based motif. Preferably,the biological persistence enhancing component enhances the biologicalhalf-life of the modified neurotoxin. More preferably, the biologicalhalf-life of the modified neurotoxin is enhanced by about 10%. Even morepreferably, the biological half-life of the modified neurotoxin isenhanced by about 100%. Generally speaking, the modified neurotoxin hasa biological persistence of about 20% to 300% more than an identicalneurotoxin without the structural modification. That is, for example,the modified neurotoxin including the biological persistence enhancingcomponent is able to cause a substantial inhibition of acetylcholinerelease from a nerve terminal for about 20% to about 300% longer than aneurotoxin that is not modified.

In a broad embodiment of the present invention, a leucine-based motif isan oligomer of seven amino acids. The oligomer is organized in to twogroups. The first five amino acids starting from the amino terminal ofthe leucine-based motif form a “quintet of amino acids.” The two aminoacids immediately following the quintet of amino acids form a “duplet ofamino acids.” In a preferred embodiment, the duplet of amino acids islocated at the carboxyl terminal region of the leucine-based motif. Inanother preferred embodiment, the quintet of amino acids includes atleast one acidic amino acid selected from a group consisting of aglutamate and an aspartate.

The duplet of amino acid includes at least one hydrophobic amino acid,for example leucine, isoleucine, methionine, alanine, phenylalanine,tryptophan, valine or tyrosine. Preferably, the duplet of amino acid isa leucine-leucine, a leucine-isoleucine, an isoleucine-leucine or anisoleucine-isoleucine. Even more preferably, the duplet is aleucine-leucine.

In one embodiment, the leucine-based motif is XDXXXLL (SEQ ID NO: 1),wherein x and may be any amino acids. In another embodiment, theleucine-based motif is XEXXXLL (SEQ ID NO: 2), wherein E is glutamicacid. In another embodiment, the duplet of amino acids may include anisoleucine or a methionine, forming XDXXXLI (SEQ ID NO: 3) or XDXXXLM(SEQ ID NO: 4), respectively. Additionally, the aspartic acid, D, may bereplaced by a glutamic acid, E, to form XEXXXLI (SEQ ID NO: 5) andXEXXXLM (SEQ ID NO: 6). In a preferred embodiment, the leucine-basedmotif is SEQ ID NO: 7.

In another embodiment, the quintet of amino acids comprises at least onehydroxyl containing amino acid, for example a serine, a threonine or atyrosine. Preferably, the hydroxyl containing amino acid can bephosphorylated. More preferably, the hydroxyl containing amino acid is aserine which can be phosphorylated to allow for the binding of adaptorproteins.

Although non-modified amino acids are provided as examples, a modifiedamino acid is also contemplated to be within the scope of thisinvention. For example, leucine-based motif may include a halogenated,preferably, fluorinated leucine.

Various leucine-based motif are found in various species. A list ofpossible leucine-based motif derived from the various species that maybe used in accordance with this invention is shown in Table 1. This listis not intended to be limiting.

TABLE 1 Species Sequence SEQ ID NO: BoNT/A FEFYKLL 7 Rat VMAT1 EEKRAIL 8Rat VMAT 2 EEKMAIL 9 Rat VAChT SERDVLL 10 Rat δ VDTQVLL 11 Mouse δAEVQALL 12 Frog γ/δ SDKQNLL 13 Chicken γ/δ SDRQNLI 14 Sheep δ ADTQVLM 15Human CD3γ SDKQTLL 16 Human CD4 SQIKRLL 17 Human δ ADTQALL 18 VMAT isvesicular monoamine transporter; VACht is vesicular acetylcholinetransporter. Italicized serine residues are potential sites ofphosphorylation.

The modified neurotoxin may be formed from any neurotoxin. Preferably,the neurotoxin used is a Clostridial neurotoxin. A Clostridialneurotoxin comprises a polypeptide having three amino acid sequenceregions. The first amino acid sequence region includes a neuronalbinding moiety which is substantially completely derived from aneurotoxin selected from a group consisting of beratti toxin; butyricumtoxin; tetani toxin; BoNT/A, B, C₁, D, E, F, and G. Preferably, thefirst amino acid sequence region is derived from the carboxyl terminalregion of a toxin heavy chain, H_(C).

The second amino acid sequence region is effective to translocate thepolypeptide or a part thereof across an endosome membrane into thecytoplasm of a neuron. In one embodiment, the second amino acid sequenceregion of the polypeptide comprises an amine terminal of a heavy chain,H_(N), derived from a neurotoxin selected from a group consisting ofberatti toxin; butyricum toxin; tetani toxin; BoNT/A, B, C₁, D, E, F,and G.

The third amino acid sequence region has therapeutic activity when it isreleased into the cytoplasm of a target cell or neuron. In oneembodiment, the third amino acid sequence region of the polypeptidecomprises a toxin light chain, L, derived from a neurotoxin selectedfrom a group consisting of beratti toxin; butyricum toxin; tetani toxin;BoNT/A, B, C₁, D, E, F, and G.

The Clostridial neurotoxin may be a hybrid neurotoxin. For example, eachof the neurotoxin's amino acid sequence regions may be derived from adifferent Clostridial neurotoxin serotype. For example, in oneembodiment, the polypeptide comprises a first amino acid sequence regionderived from the H_(C) of the tetani toxin, a second amino acid sequenceregion derived from the H_(N) of BoNt/B, and a third amino acid sequenceregion derived from the L chain of botulinum serotype E. All otherpossible combinations are included within the scope of the presentinvention.

Alternatively, all three of the amino acid sequence regions of theClostridial neurotoxin may be from the same species and same serotype.If all three amino acid sequence regions of the neurotoxin are from thesame Clostridial neurotoxin species and serotype, the neurotoxin will bereferred to by the species and serotype name. For example, a neurotoxinpolypeptide may have its first, second and third amino acid sequenceregions derived from BoNT/E. In which case, the neurotoxin is referredas BoNT/E.

Additionally, each of the three amino acid sequence regions may bemodified from the naturally occurring sequence from which they arederived. For example, the amino acid sequence region may have at leastone or more amino acid may be added or deleted as compared to thenaturally occurring sequence.

The biological persistence enhancing component, preferably theleucine-based motif, may be fused with any of the above describedneurotoxin to form a modified neurotoxin with an enhanced biologicalpersistence. “Fusing” as used in the context of this invention includescovalently adding to or covalently inserting in between a primarystructure of a neurotoxin. Preferably, the biological persistenceenhancing component is added to a Clostridial neurotoxin which does nothave a leucine-based motif in its primary structure. For example, in oneembodiment, the leucine-based motif is fused with a hybrid neurotoxin,wherein the third amino acid sequence is not derived from botulinumserotype A. In another embodiment, the leucine-based motif is fused witha BoNt/E.

In one embodiment, the leucine-based motif is fused with the third aminoacid sequence region of the neurotoxin. In a preferred embodiment, theleucine-based motif is fused with the region towards the carboxylicterminal of the third amino acid sequence region. More preferably, theleucine-based motif is fused with the carboxylic terminal of the thirdregion of a neurotoxin. Even more preferably, the leucine-based motif isfused with the carboxylic terminal of the third region of BoNt/E.

In another embodiment, the structural modification of a neurotoxin whichhas a preexisting leucine-based motif includes deleting one or moreamino acids from the leucine-based motif. Alternatively, a modifiedneurotoxin includes a structural modification which results in aneurotoxin with one or more amino acids absent from the leucine-basedmotif. The removal of one or more amino acids from the preexistingleucine-based motif is effective to reduce the biological persistence ofa modified neurotoxin. More preferably, the deletion of one or moreamino acids from the leucine-based motif of BoNT/A reduces thebiological half-life of the modified neurotoxin.

In one broad aspect of the present invention, a method is provided fortreating a biological disorder using a modified neurotoxin. Thetreatments may include treating neuromuscular disorders, autonomicnervous system disorders and pain.

The neuromuscular disorders and conditions that may be treated with amodified neurotoxin include: for example, strabismus, blepharospasm,spasmodic torticollis (cervical dystonia), oromandibular dystonia andspasmodic dysphonia (largyngeal dystonia).

For example, Borodic U.S. Pat. No. 5,053,005 discloses methods fortreating juvenile spinal curvature, i.e. scoliosis, using BoNT/A. Thedisclosure of Borodic is incorporated in its entirety herein byreference. In one embodiment, using substantially similar methods asdisclosed by Borodic, a modified neurotoxin is administered to a mammal,preferably a human, to treat spinal curvature. In a preferredembodiment, a modified neurotoxin comprising BoNT/E fused with aleucine-based motif is administered. Even more preferably, a modifiedneurotoxin comprising BoNT/E with a leucine-based motif fused to thecarboxyl terminal of its light chain is administered to the mammal,preferably a human, to treat spinal curvature. The modified neurotoxinmay be administered to treat other neuromuscular disorders using wellknown techniques that are commonly performed with BoNT/A.

Autonomic nervous system disorders may also be treated with a modifiedneurotoxin. For example, glandular malfunctioning is an autonomicnervous system disorder. Glandular malfunctioning includes excessivesweating and excessive salivation. Respiratory malfunctioning is anotherexample of an autonomic nervous system disorder. Respiratorymalfunctioning includes chronic obstructive pulmonary disease andasthma. Sanders et al. discloses methods for treating the autonomicnervous system, such as excessive sweating, excessive salivation,asthma, etc., using naturally existing botulinum toxins. The disclosureof Sander et al. is incorporated in its entirety by reference herein. Inone embodiment, substantially similar methods to that of Sanders et al.may be employed, but using a modified neurotoxin, to treat autonomicnervous system disorders such as the ones discussed above. For example,a modified neurotoxin may be locally applied to the nasal cavity of themammal in an amount sufficient to degenerate cholinergic neurons of theautonomic nervous system that control the mucous secretion in the nasalcavity.

Pain that may be treated by a modified neurotoxin include pain caused bymuscle tension, or spasm, or pain that is not associated with musclespasm. For example, Binder in U.S. Pat. No. 5,714,468 discloses thatheadache caused by vascular disturbances, muscular tension, neuralgiaand neuropathy may be treated with a naturally occurring botulinumtoxin, for example BoNT/A. The disclosures of Binder is incorporated inits entirety herein by reference. In one embodiment, substantiallysimilar methods to that of Binder may be employed, but using a modifiedneurotoxin, to treat headache, especially the ones caused by vasculardisturbances, muscular tension, neuralgia and neuropathy. Pain caused bymuscle spasm may also be treated by an administration of a modifiedneurotoxin. For example, a BoNT/E fused with a leucine-based motif,preferably at the carboxyl terminal of the BoNT/E light chain, may beadministered intramuscularly at the pain/spasm location to alleviatepain.

Furthermore, a modified neurotoxin may be administered to a mammal totreat pain that is not associated with a muscular disorder, such asspasm. In one broad embodiment, methods of the present invention totreat non-spasm related pain include central administration orperipheral administration of the modified neurotoxin.

For example, Foster et al. in U.S. Pat. No. 5,989,545 discloses that abotulinum toxin conjugated with a targeting moiety may be administeredcentrally (intrathecally) to alleviate pain. The disclosures of Fosteret al. is incorporated in its entirety by reference herein. In oneembodiment, substantially similar methods to that of Foster et al. maybe employed, but using the modified neurotoxin according to thisinvention, to treat pain. The pain to be treated may be an acute pain,or preferably, chronic pain.

An acute or chronic pain that is not associated with a muscle spasm mayalso be alleviated with a local, peripheral administration of themodified neurotoxin to an actual or a perceived pain location on themammal. In one embodiment, the modified neurotoxin is administeredsubcutaneously at or near the location of pain, for example at or near acut. In another embodiment, the modified neurotoxin is administeredintramuscularly at or near the location of pain, for example at or neara bruise location on the mammal. In another embodiment, the modifiedneurotoxin is injected directly into a joint of a mammal, for treatingor alleviating pain cause arthritis conditions. Also, frequent repeatedinjections or infusion of the modified neurotoxin to a peripheral painlocation is within the scope of the present invention. However, giventhe long lasting therapeutic effects of the present invention, frequentinjections or infusion of the neurotoxin may not be necessary. Forexample, practice of the present invention can provide an analgesiceffect, per injection, for 2 months or longer, for example 27 months, inhumans.

Without wishing to limit the invention to any mechanism or theory ofoperation, it is believed that when the modified neurotoxin isadministered locally to a peripheral location, it inhibits the releaseof neuro-substances, for example substance P, from the peripheralprimary sensory terminal. Since the release of substance P by theperipheral primary sensory terminal may cause or at least amplify paintransmission process, inhibition of its release at the peripheralprimary sensory terminal will dampen the transmission of pain signalsfrom reaching the brain.

In addition to having pharmacologic actions at the peripheral location,the modified neurotoxin of the present invention may also haveinhibitory effects in the central nervous system. Presumably theretrograde transport is via the primary afferent. This hypothesis issupported by our experimental data which shows that BoNT/A is retrogradetransported to the dorsal horn when the neurotoxin is injectedperipherally. Moreover, work by Weigand et al, Nauny-Schmiedeberg'sArch. Pharmacol. 1976; 292, 161-165, and Habermann, Nauny-Schmiedeberg'sArch. Pharmacol. 1974; 281, 47-56, showed that botulinum toxin is ableto ascend to the spinal area by retrograde transport. As such, amodified neurotoxin, for example BoNt/A with one or more amino acidsdeleted from the leucine-based motif, injected at a peripheral location,for example intramuscularly, may be retrograde transported from theperipheral primary sensory terminal to the central primary sensoryterminal.

The amount of the modified neurotoxin administered can vary widelyaccording to the particular disorder being treated, its severity andother various patient variables including size, weight, age, andresponsiveness to therapy. Generally, the dose of modified neurotoxin tobe administered will vary with the age, presenting condition and weightof the mammal, preferably a human, to be treated. The potency of themodified neurotoxin will also be considered.

Assuming a potency which is substantially equivalent to LD₅₀=2,730 U ina human patient and an average person is 75 kg, a lethal dose would beabout 36 U/kg of a modified neurotoxin. Therefore, when a modifiedneurotoxin with such an LD₅₀ is administered, it would be appropriate toadminister less than 36 U/kg of the modified neurotoxin into humansubjects. Preferably, about 0.01 U/kg to 30 U/kg of the modifiedneurotoxin is administered. More preferably, about 1 U/kg to about 15U/kg of the modified neurotoxin is administered. Even more preferably,about 5 U/kg to about 10 U/kg modified neurotoxin is administered.Generally, the modified neurotoxin will be administered as a compositionat a dosage that is proportionally equivalent to about 2.5 cc/100 U.Those of ordinary skill in the art will know, or can readily ascertain,how to adjust these dosages for neurotoxin of greater or lesser potency.

Although examples of routes of administration and dosages are provided,the appropriate route of administration and dosage are generallydetermined on a case by case basis by the attending physician. Suchdeterminations are routine to one of ordinary skill in the art (see forexample, Harrison's Principles of Internal Medicine (1998), edited byAnthony Fauci et al., 14^(th) edition, published by McGraw Hill). Forexample, the route and dosage for administration of a modifiedneurotoxin according to the present disclosed invention can be selectedbased upon criteria such as the solubility characteristics of themodified neurotoxin chosen as well as the types of disorder beingtreated.

The modified neurotoxin may be produced by chemically linking theleucine-based motif to a neurotoxin using conventional chemical methodswell known in the art. The neurotoxin may be obtained from a harvestingneurotoxins. For example, BoNt/E can be obtained by establishing andgrowing cultures of Clostridium botulinum in a fermenter and thenharvesting and purifying the fermented mixture in accordance with knownprocedures. All the botulinum toxin serotypes are initially synthesizedas inactive single chain proteins which must be cleaved or nicked byproteases to become neuroactive. The bacterial strains that makebotulinum toxin serotypes A and G possess endogenous proteases andserotypes A and G can therefore be recovered from bacterial cultures inpredominantly their active form. In contrast, botulinum toxin serotypesC₁, D and E are synthesized by nonproteolytic strains and are thereforetypically unactivated when recovered from culture. Serotypes B and F areproduced by both proteolytic and nonproteolytic strains and thereforecan be recovered in either the active or inactive form. However, eventhe proteolytic strains that produce, for example, the BoNt/B serotypeonly cleave a portion of the toxin produced. The exact proportion ofnicked to unnicked molecules depends on the length of incubation and thetemperature of the culture. Therefore, a certain percentage of anypreparation of, for example, the BoNt/B toxin is likely to be inactive,possibly accounting for the known significantly lower potency of BoNt/Bas compared to BoNt/A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that BoNt/B has, upon intramuscular injection,a shorter duration of activity and is also less potent than BoNt/A atthe same dose level.

The modified neurotoxin may also be produced by recombinant techniques.Recombinant techniques are preferable for producing a neurotoxin havingamino acid sequence regions from different Clostridial species or havingmodified amino acid sequence regions. Also, the recombinant technique ispreferable in producing BoNT/A with the leucine-based motif beingmodified by deletion. The technique includes steps of obtaining geneticmaterials from natural sources, or synthetic sources, which have codesfor a neuronal binding moiety, an amino acid sequence effective totranslocate the neurotoxin or a part thereof, and an amino acid sequencehaving therapeutic activity when released into a cytoplasm of a targetcell, preferably a neuron. In a preferred embodiment, the geneticmaterials have codes for the biological persistence enhancing component,preferably the leucine-based motif, the H_(C), the H_(N) and the L chainof the Clostridial neurotoxins and fragments thereof. The geneticconstructs are incorporated into host cells for amplification by firstfusing the genetic constructs with a cloning vectors, such as phages orplasmids. Then the cloning vectors are inserted into hosts, preferablyE. coli's. Following the expressions of the recombinant genes in hostcells, the resultant proteins can be isolated using conventionaltechniques.

There are many advantages to producing these modified neurotoxinsrecombinantly. For example, to form a modified neurotoxin, a modifyingfragment must be attached or inserted into a neurotoxin. The productionof neurotoxin from anaerobic Clostridium cultures is a cumbersome andtime-consuming process including a multi-step purification protocolinvolving several protein precipitation steps and either prolonged andrepeated crystallization of the toxin or several stages of columnchromatography. Significantly, the high toxicity of the product dictatesthat the procedure must be performed under strict containment (BL-3).During the fermentation process, the folded single-chain neurotoxins areactivated by endogenous clostridial proteases through a process termednicking to create a dichain. Sometimes, the process of nicking involvesthe removal of approximately 10 amino acid residues from thesingle-chain to create the dichain form in which the two chains remaincovalently linked through the intrachain disulfide bond.

The nicked neurotoxin is much more active than the unnicked form. Theamount and precise location of nicking varies with the serotypes of thebacteria producing the toxin. The differences in single-chain neurotoxinactivation and, hence, the yield of nicked toxin, are due to variationsin the serotype and amounts of proteolytic activity produced by a givenstrain. For example, greater than 99% of Clostridial botulinum serotypeA single-chain neurotoxin is activated by the Hall A Clostridialbotulinum strain, whereas serotype B and E strains produce toxins withlower amounts of activation (0 to 75% depending upon the fermentationtime). Thus, the high toxicity of the mature neurotoxin plays a majorpart in the commercial manufacture of neurotoxins as therapeutic agents.

The degree of activation of engineered clostridial toxins is, therefore,an important consideration for manufacture of these materials. It wouldbe a major advantage if neurotoxins such as botulinum toxin and tetanustoxin could be expressed, recombinantly, in high yield inrapidly-growing bacteria (such as heterologous E. coli cells) asrelatively non-toxic single-chains (or single chains having reducedtoxic activity) which are safe, easy to isolate and simple to convert tothe fully-active form.

With safety being a prime concern, previous work has concentrated on theexpression in E. coli and purification of individual H and L chains oftetanus and botulinum toxins; these isolated chains are, by themselves,non-toxic; see Li et al., Biochemistry 33:7014-7020 (1994); Zhou et al.,Biochemistry 34:15175-15181 (1995), hereby incorporated by referenceherein. Following the separate production of these peptide chains andunder strictly controlled conditions the H and L chains can be combinedby oxidative disulphide linkage to form the neuroparalytic di-chains.

EXAMPLES

The following non-limiting examples provide those of ordinary skill inthe art with specific preferred methods to treat non-spasm related painwithin the scope of the present invention and are not intended to limitthe scope of the invention.

Example 1 Treatment of Pain Associated with Muscle Disorder

An unfortunate 36 year old woman has a 15 year history oftemporomandibular joint disease and chronic pain along the masseter andtemporalis muscles. Fifteen years prior to evaluation she notedincreased immobility of the jaw associated with pain and jaw opening andclosing and tenderness along each side of her face. The left side isoriginally thought to be worse than the right. She is diagnosed ashaving temporomandibular joint (TMJ) dysfunction with subluxation of thejoint and is treated with surgical orthoplasty meniscusectomy andcondyle resection.

She continues to have difficulty with opening and closing her jaw afterthe surgical procedures and for this reason, several years later, asurgical procedure to replace prosthetic joints on both sides isperformed. After the surgical procedure progressive spasms and deviationof the jaw ensues. Further surgical revision is performed subsequent tothe original operation to correct prosthetic joint loosening. The jawcontinues to exhibit considerable pain and immobility after thesesurgical procedures. The TMJ remained tender as well as the muscleitself. There are tender points over the temporomandibular joint as wellas increased tone in the entire muscle. She is diagnosed as havingpost-surgical myofascial pain syndrome and is injected with 7 U/kg ofthe modified neurotoxin into the masseter and temporalis muscles,preferably the modified neurotoxin is BoNT/E fused with a leucine-basedmotif.

Several days after the injections she noted substantial improvement inher pain and reports that her jaw feels looser. This gradually improvesover a 2 to 3 week period in which she notes increased ability to openthe jaw and diminishing pain. The patient states that the pain is betterthan at any time in the last 4 years. The improved condition persistsfor up to 27 months after the original injection of the modifiedneurotoxin.

Example 2 Treatment of Pain Subsequent to Spinal Cord Injury

A patient, age 39, experiencing pain subsequent to spinal cord injury istreated by intrathecal administration, for example by spinal tap or bycatherization (for infusion), to the spinal cord, with between about 0.1U/kg of the modified neurotoxin, preferably the modified neurotoxin isBoNT/E fused with a leucine-based motif. The particular toxin dose andsite of injection, as well as the frequency of toxin administrationsdepend upon a variety of factors within the skill of the treatingphysician, as previously set forth. Within about 1 to about 7 days afterthe modified neurotoxin administration, the patient's pain issubstantially reduced. The pain alleviation persists for up to 27months.

Example 3 Peripheral Administration of a Modified Neurotoxin to Treat“Shoulder-Hand Syndrome”

Pain in the shoulder, arm, and hand can develop, with musculardystrophy, osteoporosis, and fixation of joints. While most common aftercoronary insufficiency, this syndrome may occur with cervicalosteoarthritis or localized shoulder disease, or after any prolongedillness that requires the patient to remain in bed.

A 46 year old woman presents a shoulder-hand syndrome type pain. Thepain is particularly localized at the deltoid region. The patient istreated by a bolus injection of between about 0.05 U/kg to about 2 U/kgof a modified neurotoxin subcutaneously to the shoulder, preferably themodified neurotoxin is BoNT/E fused with a leucine-based motif. Theparticular dose as well as the frequency of administrations depends upona variety of factors within the skill of the treating physician, aspreviously set forth. Within 1-7 days after modified neurotoxinadministration the patient's pain is substantially alleviated. Theduration of the pain alleviation is from about 7 to about 27 months.

Example 4 Peripheral Administration of a Modified Neurotoxin to TreatPostherpetic Neuralgia

Postherpetic neuralgia is one of the most intractable of chronic painproblems. Patients suffering this excruciatingly painful process oftenare elderly, have debilitating disease, and are not suitable for majorinterventional procedures. The diagnosis is readily made by theappearance of the healed lesions of herpes and by the patient's history.The pain is intense and emotionally distressing. Postherpetic neuralgiamay occur any where, but is most often in the thorax.

A 76 year old man presents a postherpetic type pain. The pain islocalized to the abdomen region. The patient is treated by a bolusinjection of between about 0.05 U/kg to about 2 U/kg of a modifiedneurotoxin intradermally to the abdomen, preferably the modifiedneurotoxin is BoNT/E fused with a leucine-based motif. The particulardose as well as the frequency of administrations depends upon a varietyof factors within the skill of the treating physician, as previously setforth. Within 1-7 days after modified neurotoxin administration thepatient's pain is substantially alleviated. The duration of the painalleviation is from about 7 to about 27 months.

Example 5 Peripheral Administration of a Modified Neurotoxin to TreatNasopharyngeal Tumor Pain

These tumors, most often squamous cell carcinomas, are usually in thefossa of Rosenmuller and may invade the base of the skull. Pain in theface is common. It is constant, dull-aching in nature.

A 35 year old man presents a nasopharyngeal tumor type pain. Pain isfound at the lower left cheek. The patient is treated by a bolusinjection of between about 0.05 U/kg to about 2 U/kg of a modifiedneurotoxin intramuscularly to the cheek, preferably the modifiedneurotoxin is BoNT/E fused with a leucine-based motif. The particulardose as well as the frequency of administrations depends upon a varietyof factors within the skill of the treating physician, as previously setforth. Within 1-7 days after modified neurotoxin administration thepatient's pain is substantially alleviated. The duration of the painalleviation is from about 7 to about 27 months.

Example 6 Peripheral Administration of a Modified Neurotoxin to TreatInflammatory Pain

A patient, age 45, presents an inflammatory pain in the chest region.The patient is treated by a bolus injection of between about 0.05 U/kgto about 2 U/kg of a modified neurotoxin intramuscularly to the chest,preferably the modified neurotoxin is BoNT/E fused with a leucine-basedmotif. The particular dose as well as the frequency of administrationsdepends upon a variety of factors within the skill of the treatingphysician, as previously set forth. Within 1-7 days after modifiedneurotoxin administration the patient's pain is substantiallyalleviated. The duration of the pain alleviation is from about 7 toabout 27 months.

Example 7 Treatment of Excessive Sweating

A male, age 65, with excessive unilateral sweating is treated byadministering 0.05 U/kg to about 2 U/kg of a modified neurotoxin,depending upon degree of desired effect. Preferably the modifiedneurotoxin is BoNT/E fused with a leucine-based motif. Theadministration is to the gland nerve plexus, ganglion, spinal cord orcentral nervous system. The specific site of administration is to bedetermined by the physician's knowledge of the anatomy and physiology ofthe target glands and secretary cells. In addition, the appropriatespinal cord level or brain area can be injected with the toxin. Thecessation of excessive sweating after the modified neurotoxin treatmentis up to 27 months.

Example 8 Post Surgical Treatments

A female, age 22, presents a torn shoulder tendon and undergoesorthopedic surgery to repair the tendon. After the surgery, the patientis administered intramuscularly with about 0.05 U/kg to about 2 U/kg ofa modified neurotoxin to the shoulder. Preferably, the modifiedneurotoxin is a BoNT/A wherein the leucine-based motif is removed. Thespecific site of administration is to be determined by the physician'sknowledge of the anatomy and physiology of the muscles. The administeredmodified neurotoxin reduces movement of the arm to facilitate therecovery from the surgery. The effect of the modified neurotoxin is forabout five weeks.

Example 9 Production of a Modified Neurotoxin with an EnhancedBiological Persistence

A modified neurotoxin may be produced by employing recombinanttechniques in conjunction with conventional chemical techniques.

The neurotoxin that is to be fused with the leucine-based motif to forma modified neurotoxin may be produced recombinantly. The recombinanttechnique includes steps of obtaining genetic materials from either DNAcloned from natural sources, or synthetic oligonucleotide sequences,which have codes for a neurotoxin, preferably BoNT/E. The geneticconstructs are incorporated into host cells for amplification by firstfusing the genetic constructs with a cloning vectors, such as phages orplasmids. Then the cloning vectors are inserted into hosts, preferablyE. coli's. Following the expressions of the recombinant genes in hostcells, the resultant proteins can be isolated using conventionaltechniques.

The neurotoxin, preferably BoNT/E, derived from the recombinanttechniques can then be covalently fused with a leucine-based motif.Preferably, the leucine-based motif is fused to the light chain ofBoNT/E at the carboxyl terminal. The fusion of the leucine-based motifwith BoNT/E is achieved via chemical coupling using reagents andtechniques known to those skilled in the art, for example PDPH/EDAC andTraut's reagent chemistry.

The modified neurotoxin produced according to this example has anenhanced biological persistence. Preferably, the biological persistenceis enhanced by about 20% to about 300% relative to an identicalneurotoxin without a leucine-based motif.

Example 10 Production of a Modified Neurotoxin with a Reduced BiologicalPersistence

A modified neurotoxin with a reduced biological persistence may beproduced by employing recombinant techniques. The recombinant techniqueincludes steps of obtaining genetic materials from a syntheticoligonucleotide sequences, which have codes for a neurotoxin, preferablyBoNT/A, which does not have genetic codings for a leucine-based motif.The genetic constructs are incorporated into host cells foramplification by first fusing the genetic constructs with a cloningvectors, such as phages or plasmids. Then the cloning vectors areinserted into hosts, preferably E. coli's. Following the expressions ofthe recombinant genes in host cells, the resultant proteins can beisolated using conventional techniques.

The modified neurotoxin produced according to this example has a reducedbiological persistence. Preferably, the biological persistence isreduced by about 20% to about 300% relative to an identical neurotoxin,for example BoNT/A, with the leucine-based motif.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of modified neurotoxins can be effectivelyused in the methods of the present invention in place of clostridialneurotoxins. Also, the corresponding genetic codes, i.e. DNA sequence,to the modified neurotoxins are also considered to be part of thisinvention. Additionally, the present invention includes peripheraladministration methods wherein two or more modified neurotoxins, forexample BoNT/E with a fused leucine-based motif and BoNT/B with fusedleucine-based motif, are administered concurrently or consecutively.While this invention has been described with respect to various specificexamples and embodiments, it is to be understood that the invention isnot limited thereto and that it can be variously practiced with thescope of the following claims.

1. A modified botulinum neurotoxin type E having increased biologicalhalf-life, wherein the modification is the addition of the leucine-basedmotif of SEQ ID NO: 3, and wherein the added leucine-based motifincreases biological half-life of the modified botulinum toxin type Erelative to an identical botulinum toxin type E without the addedleucine-based motif.
 2. The modified botulinum neurotoxin type E ofclaim 1, wherein the added leucine-based motif is SEQ ID NO:
 14. 3. Abotulinum neurotoxin type E comprising a modification, wherein themodification is the addition of the leucine-based motif of SEQ ID NO: 3,and wherein the added leucine-based motif increases biological half-lifeof the botulinum toxin type E relative to an identical botulinum toxintype E without the added leucine-based motif.
 4. The modified botulinumneurotoxin type E of claim 3, wherein the added leucine-based motif isSEQ ID NO:
 14. 5. A botulinum neurotoxin type E comprising amodification, wherein the modification is the addition of theleucine-based motif of SEQ ID NO: 2, and wherein the added leucine-basedmotif increases biological half-life of the botulinum toxin type Erelative to an identical botulinum toxin type E without the addedleucine-based motif.
 6. The botulinum neurotoxin type E of claim 5,wherein the added leucine-based motif is SEQ ID NO: 7, SEQ ID NO: 10 orSEQ ID NO: 12.